Inflammation plays a major role in breast cancer development and progression. Cyclooxygenase (COX)-2 and its product prostaglandin E2 (PGE2) are major mediators of inflammation in cancer cells. In this competitive renewal application, our new directions are built on discoveries made during the previous funding period. With imaging we uncovered the role of COX-2 in mechanotransduction, its impact on attracting cancer associated fibroblasts (CAFs), altering collagen fiber structure and function, and increasing metastasis. We found that COX-2 overexpression in tumors impacted tumor and spleen metabolism that opens the possibility of spleen dysregulation contributing to tumors escaping immune surveillance. In our efforts to downregulate COX-2 in tumors with siRNA delivery, we found that most nanoparticles (NPs) significantly increased COX-2, until we developed a biocompatible translatable dextran NP that successfully delivered COX-2 siRNA to effectively decrease COX-2 and PGE2 in cancer cells and tumors. These observations have resulted in our three new aims that focus on advancing our understanding COX-2 in cancer, and in developing translatable strategies to target COX-2 and CAFs. In Aim 1 will investigate the role of COX-2 in altering extracellular matrix (ECM) stiffness and composition, and the impact of eliminating CAFs that are a source of COX-2, on the ECM and on tumor immune cells and metastasis. In Aim 2 we will identify the causes of the metabolic changes in the tumor and spleen caused by COX-2, and the effects of eliminating CAFs on tumor and spleen metabolism, and splenocytes. In Aim 3 we will downregulate COX-2 in established tumors with siRNA and determine the impact on the ECM, metabolism, and immune cells. A small component of this Aim will include developing cytokine scavenging NPs that are based on the modified dextran NP we developed. We will evaluate the ability of these NPs to reduce cytokines in tumor interstitial fluid. The studies will be performed with triple negative breast cancer models genetically engineered to overexpress or downregulate COX-2 in immune suppressed and immune competent mice. These studies may lead to ECM modification strategies, including CAF elimination, to alter ECM stiffness to reduce cancer aggressiveness, and may result in new metabolic targets and biomarkers for immunotherapy, as well as new strategies to convert an immune suppressive tumor microenvironment into an immune active one. !